The eNOS production is significantly lower in hCB-ECs as compared to HUVECs when seeded on NT scaffolds (* 0.05; = 4). (2008) [33], with minor modifications. Briefly, cord blood (20C100 mL) was diluted 1:1 with Hanks balanced salt solution (HBSS), and then overlaid onto an equivalent volume of Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA). To isolate mononuclear cells, the diluted cord blood was centrifuged for 30 min at room temperature at 740 = 6 scaffolds each) were imaged using a JEOL JSM-6335F scanning electron microscope (JEOL USA, Peabody, MA, USA) at 3 kV at a magnification of 10,000. The SEM images were binarized and the porosity was calculated as the ratio of the total number of fiber pixels to the total number of pixels in the image. The fiber diameter was calculated by manually measuring the diameter of 120 randomly selected fibers per scaffold treatment via freehand lines superimposed over the SEM images in ImageJ. Multiphoton microscopy was used for the A-9758 three-dimensional imaging of our electrospun scaffolds. The Pitt Advanced Intravital Microscope (AIM) for multiphoton imaging at the University of Pittsburgh Soft Tissue Biomechanics Laboratory allowed us to measure the change in scaffold thickness. This Olympus BX51 upright laser scanning microscope (Olympus, Tokyo, Japan) was coupled to a 120-fs tunable pulsed Titanium-Sapphire laser (Coherent Inc, Santa Clara, CA, USA) and an Olympus XLUMPLFL 20 water immersion objective with a numerical aperture of 0.9 [40,41]. The fibers were imaged centering the laser at 780 nm to excite the autofluorescence signal from the scaffolds (NADH), split with a 568 nm dichroic mirror, A-9758 and collected through a 525/50 nm bandpass filter. The signal was collected over a 400 m 400 m field of view at 2-m z-step-size along the scaffold thickness. 2.5. Effect of Surface Modification on Cell Growth hCB-ECs and HUVECs were seeded in NT and TC scaffolds at 10,000 cells/scaffold and cultured for 7 days. The culture medium was changed every other day and cultures were maintained in a humidified environment at 37 C and 5% CO2. Cell growth was evaluated after 7 days of culture. A sample of approximately 25 mm2 was cut from each scaffold, and cell number was measured by MTS assay. Briefly, cell-seeded scaffolds were incubated in culture medium supplemented with CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) at 37 C for 4 h. Supernatant was collected and the absorbance at 490 nm was recorded. Background absorbance from the NT and TC scaffolds was obtained from nonseeded scaffolds. Cell number was calculated Mouse monoclonal to CD23. The CD23 antigen is the low affinity IgE Fc receptor, which is a 49 kDa protein with 38 and 28 kDa fragments. It is expressed on most mature, conventional B cells and can also be found on the surface of T cells, macrophages, platelets and EBV transformed B lymphoblasts. Expression of CD23 has been detected in neoplastic cells from cases of B cell chronic Lymphocytic leukemia. CD23 is expressed by B cells in the follicular mantle but not by proliferating germinal centre cells. CD23 is also expressed by eosinophils. based on our calibration curves (Figure S3). For cell imaging, the scaffolds were fixed with 2% formaldehyde and stained with Alexa Fluor? 568 phalloidin (Life Technologies, Carlsbad, CA, USA) to visualize f-actin following the manufacturers instructions. To stain the nuclei, the scaffolds were treated for 24 h with VECTASHIELD? DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA). The Pitt AIM with a 20 water immersion objective was used to visualize the cells growing in the scaffolds along the scaffold depth. The nuclei (blue), fibers (green), and f-actin (red) were imaged simultaneously and colocalized using three different photomultiplier tubes (PMTs). The laser was centered at = 780 nm to excite simultaneously DAPI, the autofluorescence signal from the scaffolds (NADH), and Alexa Fluor? A-9758 568. In.